Dissection of the translocation and chaperoning functions of yeast BiP/Kar2p in vivo.
We used the rat nerve growth factor receptor ectodomain (NGFRe) and Escherichia coli ss-lactamase to dissect the functions of Saccharomyces cerevisiae BiP/Kar2p in vivo. Both were fused to the Hsp150Delta-polypeptide, which promotes proper folding of heterologous proteins which otherwise are misfolded in the yeast ER. Hsp150Delta-NGFRe and Hsp150Delta-beta-lactamase acquired disulfides and were properly folded and ONcreted to the culture medium. When disulfide formation was prevented by incubating cells with dithiothreitol (DTT), Hsp150Delta-NGFRe remained in the endoplasmic reticulum (ER). The occupancy of an otherwise partially used N-glycosylation site of reduced NGFRe was complete suggesting that, normally, folding and disulfide formation occurred as rapidly as N-glycosylation. Removal of DTT resulted in remarkably rapid disulfide formation and secretion, suggesting only mild conformational distortion of reduced NGFRe. In contrast, reduced Hsp150(Delta)-ss-lactamase was severely misfolded and attained a secretion competent conformation more slowly after reoxidation. When kar2-159 cells were incubated at permissive temperature 24 degrees C with DTT, the reporter proteins were retained in the ER. After shift of the cells to 34 degrees C to inactivate BiP/Kar2p irreversibly, and subsequent removal of DTT, most pre-accumulated Hsp150Delta-NGFRe was rapidly secreted, whereas Hsp150Delta-beta-lactamase was secretion incompetent. Thus, Hsp150Delta-NGFRe did not require BiP/Kar2p for conformational maturation, though translocation was dependent on BiP/Kar2p. Apparently proteins differ in their post-translocational requirements for BiP/Kar2p, indicating that translocation and chaperoning are distinct functions.
Holkeri
H
H
Institute of Biotechnology, University of Helsinki, Finland.
Paunola
E
E
Jämsä
E
E
Makarow
M
M
D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D004220:Disulfides / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000378:metabolism / Q000502:physiology*; D006023:Glycoproteins; D006031:Glycosylation / Q000187:drug effects; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism / Q000502:physiology*; D006360:Heat-Shock Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000378:metabolism / Q000502:physiology*; D011487:Protein Conformation; D051381:Rats; D017475:Receptors, Nerve Growth Factor / Q000187:drug effects / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000187:drug effects / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins
D004220:Disulfides; D005656:Fungal Proteins; D006023:Glycoproteins; C074229:HSP150 protein, S cerevisiae; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; C060153:KAR2 protein, yeast; D018832:Molecular Chaperones; D017475:Receptors, Nerve Growth Factor; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D004229:Dithiothreitol